1. Field of the Invention
The present invention relates to the field of post-translational modification of proteins, and more particularly, to an enzyme catalyzing carboxyl methylation of signaling molecules and DNA encoding same. The present invention also relates to a method of screening for inhibitors of carboxyl methylation, which inhibitors may serve as therapeutic agents in the treatment of inflammation and cancer.
2. Description of the Related Art
Proteins that are posttranslationally modified by prenylation (farnesylation or geranylgeranylation) of a C-terminal cysteine are usually further modified by proteolysis and .alpha.-carboxyl methylation. There are two major classes of proteins that are prenylated on cysteine residues. The first major class of these prenylated proteins are those which end with the sequence CysXaaXaaXaa (SEQ ID NO:6), hereinafter referred to as CXXX, indicating that the primary translation product has a cysteine four residues from the C-terminus. Proteins of this class are often referred to as CAAX proteins, where A=aliphatic amino acid and X=any amino acid, because most, but not all, such proteins have aliphatic residues at the indicated positions (Clarke, S., 1992).
Among the large number of proteins ending with the CXXX consensus signal for prenylation are yeast mating pheromones, nuclear lamins, members of ras and Rho families of the Ras superfamily of GTPases, and the .gamma. subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins.
In most cases, the final amino acid of the CXXX sequence determines whether the primary translation product is farnesylated or geranylgeranylated on the C-terminal cysteine residue by two related (type I) but distinct prenyltransferases. If the final amino acid of the C-terminus is leucine or phenylalanine, the primary translation product is geranylgeranylated (Finegold et al., 1991; Kinsella et al., 1992). Other amino acids, such as serine, methionine, or glutamine, at the final amino acid position, causes the primary translation product to be farnesylated. Thus, cytosolic type I prenyltransferases recognize the CXXX consensus sequence and catalyze the attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl polyisoprene chain via a thioether linkage to the cysteine residue (Glomset et al., 1990; Maltese, W. A., 1990; Seabra et al., 1991).
Once prenylated, these proteins become substrates for a protease that removes the XXX sequence (final three amino acids at the C-terminus), leaving the prenylcysteine as the new C-terminus. It is this C-terminal prenylcysteine moiety that then becomes a substrate for prenylcysteine carboxyl methyltransferase, which methylesterifies the .alpha. carboxyl group (Clarke et al., 1988; Stephenson et al., 1990). Unlike prenylation and proteolysis, however, carboxyl methylation is reversible under physiologic conditions (Venkatasubramanian et al., 1980; Chelsky et al., 1985). FIG. 1 schematically illustrates the series of post-translational modifications of proteins having the C-terminal CXXX consensus sequence using a Rho-protein as an example.
The other major class of prenylated proteins is the rab family of ras-related GTPases which end with the sequence CysXaaCys or CysCys. One or both of the C-terminal cysteine residues are geranylgeranylated by a distinct type II geranylgeranyl transferase. The C-terminal geranylgeranylated cysteine residues of rab proteins are also substrates for prenylcysteine carboxyl methyltransferase.
Yeast mating pheromones absolutely require carboxyl methylation for their interaction with receptors. The gene product of the wild-type Ste14 gene characterized and sequenced in Saccharomyces cerevisiae was identified to be the farnesylcysteine C-terminal carboxyl methyltransferase which mediates the C-terminal methylation of the yeast a-factor pheromone and the ras proteins of S. cerevisiae (Hrycyna et al., 1990 and 1991; Ashby et al., 1993). In human neutrophils, the laboratory of the present inventor reported that prenylcysteine carboxyl methylation of Rho GTPases is stimulated by inflammatory agonists and that agents that block prenylcysteine carboxyl methyltransferase inhibit neutrophil signal transduction, (Philips et al., 1993). Thus, inhibitors of prenylcysteine carboxyl methyl transferase are expected to serve a therapeutic role as antiinflammatory agents.
The family of Ras proteins which are prenylated play a central role in the regulation of cell growth and integration of the regulatory signals which govern the cell cycle and proliferation. It is now known that there are four alleles of ras, H-ras, K-ras (both exon 4a and 4b splice variants), and N-ras, in mammalian cells. Mutants of these ras genes were among the first oncogenes to be identified for their ability to transform cells to a cancerous phenotype (Barbacid, 1987), and mutations of the ras genes (H-ras, K-ras, and N-ras) have been demonstrated to be associated with unregulated cell proliferation and are found in an estimated 30% of all human cancers (Rodenhuis, 1992).
The function of normal and oncogenic Ras proteins is absolutely dependent on the series of posttranslational modifications (see FIG. 1). Indeed, such posttranslational modifications are necessary for both membrane targeting and localization. Because Ras function is dependent on the localization to the plasma membrane and association with the plasma membrane, and because constitutively active mutant Ras proteins and several other prenylated Ras-related GTPases have the capacity to transform cells, it has been recognized that each of the enzymatic posttranslational modification steps are new drug targets (Gibbs, 1991). There has been much activity in developing inhibitors of prenyltransferases, which catalyze the first step of the posttranslational modifications, to block the membrane targeting and localization of the Ras protein (Koblan et al., 1996). Tests have shown that prenyltransferase inhibitors block the maturation of the Ras protein and reverse the cancerous transformation induced by mutant ras genes in cell culture and that the formation of new tumors by abnormal Ras proteins was prevented in animals. Furthermore, the farnesyl transferase inhibitors appear to be quite specific and do not affect normal cells (Koblan et al., 1996; Gibbs et al., 1996; Oliff et al., 1996).
It is expected that inhibitors that target and block prenylcysteine carboxyl methyltransferase, the third step of the posttranslational modification, will also serve as a therapeutic in cancer treatment as well as in other hyperproliferative disorders, such as psoriasis, precancer, etc. Prenylcysteine carboxyl methyltransferases, however, have defied biochemical purification because they cannot be extracted from biological membranes in an active state. Thus, no tool useful in identifying such inhibitors has existed in the prior art.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to the applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.